Biotin is an essential vitamin used as a cofactor in carboxylation reactions central to human metabolism, particularly in enzymes involved in fatty acid biosynthesis, gluconeogenesis, and branched-chain amino acid catabolism. Biotin is biosynthesized in microbes and plants through a four-step pathway that terminates with addition of sulfur and formation of the thiophane ring. This sulfur insertion is an impressive feat of enzyme catalysis that requires removal of two unactivated hydrogen atoms from the substrate dethiobiotin. In E. coli, sulfur insertion is catalyzed by the homodimeric iron-sulfur protein biotin synthase (BS), requires flavodoxin and S-adenosylmethionine (AdoMet), and produces methionine and 5'-deoxyadenosine. Collectively these traits indicate that biotin synthase is an AdoMet-dependent radical enzyme that catalyzes reductive cleavage of the AdoMet sulfonium to produce 5'-deoxyadenosyl radicals. We have proposed and provided evidence for a mechanism in which AdoMet coordinates a [4Fe-4S] 2+ cluster and subsequent electron transfer into the AdoMet/[4Fe-4S] 2+ cluster complex results in production of a 5'-deoxyadenosyl radical. This radical abstracts a hydrogen atom from the substrate, dethiobiotin, generating a substrate carbon radical. The substrate radical is quenched by a sulfur atom from the [2Fe-2S] 2+ cluster, generating 9-mercaptodethiobiotin as a discrete intermediate. A second AdoMet cleavage triggers a similar reaction sequence, leading to formation of the second C-S bond in the product biotin. We have solved the crystal structure of E. coil biotin synthase with substrates bound and the relative positions of the respective reactants strongly supports this mechanistic proposal. Armed with knowledge derived from the structure, along with the expertise we have developed in anaerobic biochemistry, we are poised to test several key aspects of this mechanism. First, we will use the structure as a guide and test the roles of several conserved protein residues in catalysis. Second, we have developed a half-turnover reaction that will allow us to study the formation of 9-mercaptodethiobiotin and the associated iron-sulfur cluster states using stopped-flow and quench-flow kinetic methods. Finally, since the mechanism we propose describes a single turnover that results in destruction of the [2Fe-2S] 2+ cluster, we are examining possible mechanisms for cluster reassembly that facilitate multiple enzyme turnovers in vivo. A detailed knowledge of the chemical requirements for production of 5'-deoxyadenosyl radicals and for controlled utilization of these radicals for substrate activation and biotin formation will contribute to our expanding understanding of the general features common to all radical enzymes.